advancing science with dna sequence metatranscriptomics: challenges and progress shaomei he doe...

Advancing Science with DNA Sequence

Metatranscriptomics:Challenges and Progress

Shaomei HeDOE Joint Genome Institute

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Metatranscriptomics

Metatranscriptome

The complete collection of transcribed sequences in a microbial community:

Protein-coding RNA (mRNA) Non-coding RNA (rRNA, tRNA, regulatory RNA, etc)

Metatranscriptomics studies: Community functions Response to different

environments Regulation of gene expression


Evolving of Metatranscriptomics

cDNA clone libraries + Sanger sequencing

Microarrays

RNA-seq enabled by next-generation sequencing technologies.

Sorek & Cossart, NRG (2010) 11, 9-16

RNA-seq is superior to microarrays in many ways in microbial community gene expression analysis.


Challenges in Metatranscriptomics

Wet lab Low RNA yield from environmental samples Instability of RNA (half-lives on the order of

minutes) High rRNA content in total RNA (mRNA

accounts for 1-5% of total RNA)

http://cybernetnews.com/vista-recovery-disc/

http://www.nwfsc.noaa.gov/index.cfm

Bioinformatics General challenges with short reads and large

data size Small overlap between metagenome and

metatranscriptome, or complete lack of metagenome reference


rRNA Removal Methods

Method rRNA feature usedInput RNA

Manipulate raw RNA

Before cDNA synthesis

Subtractive hybridization Conserved sequence

HighYes

RNase H digestion

Exonuclease digestion 5’ monophosphate

Gel extraction Size

Biased poly(A) tailing 2o structure Low

During cDNA synthesis

Not-so-random primers Sequence feature Low No

After cDNA synthesis

Library normalization w/ DSN High abundance Low No


Validation of two rRNA removal kits

Hyb Exo

Capture Oligo

Magnetic Bead

rRNA

mRNA

Subtractive Hybridization

MICROBExpress Bacterial mRNA Enrichment(Ambion)

Exonuclease Digestion

mRNA-ONLY Prokaryotic mRNA Isolation(Epicentre)

5’ Monophosphate Dependent Exonuclease

rRNA

mRNA

5’ P

5’ PPP


Objectives

Validate the performance of Hyb and Exo kits on

“synthetic” microbial communities, using Illumina

sequencing to evaluate:

Efficiency of rRNA removal

Fidelity of mRNA relative transcript abundance

Hyb 2 x Hyb Exo Hyb + Exo Exo + Hyb

Treatments:


What we learned

rRNA removal efficiency for both kits was community composition and RNA integrity dependent.

Exo degraded some mRNA, introducing larger variation than Hyb.

Combining Hyb and Exo provided higher rRNA removal than used alone, but the fidelity was significantly compromised.

Hyb had high fidelity, but its performance was limited by rRNA probe target range and RNA integrity.


Customized subtractive hybridization

Stewart et al, ISME J (2010) 4, 896–907

Customized probes specific to communities of interest

Probes cover near-full-length rRNA, and should also capture partially degraded (fragmented) rRNA

It has been applied on marine metatranscriptome samples to substantially reduce rRNA.


Duplex-specific nuclease (DSN)

• Efficient on E. coli (final rRNA% = 26 ± 11%)• Preserved mRNA relative abundance• Little reduction of the very abundant mRNA

Total RNA

RNA-seq library construction

Library normalization using DSN

Denature ds-DNA at high temp

Re-anneal to ds-DNA at lower temp.

DSN degrades DNA duplex which is presumably from abundant transcripts.

Yi et al, Nucleic Acids Res, 2011, 1-9


Still efficient and “faithful” for microbial communities?

0

0.5

1

1.5

2

2.5

3

1 101 201 301 401 501 601 701 801 901 1001

Rank of OTU

Rel

ativ

e ab

un

dan

ce o

f O

TU

(%

)

Environmental microbial communities are very diverse, with a long tail of minor community members.

Typical species rank abundance


Epicentre Ribo-Zero Epicentre Ribo-Zero TM TM Kit Kit

- Cindi Hoover, JGI

Another subtractive hybridization-based kit.

High fidelity!


Test on a real sample from cow rumenTest on a real sample from cow rumen

Effective even on complex metatranscriptome samples.

Sample % rRNA % Map (rumen metagenome)

% Other

No depletion control

82.4 3.4 10.5

Ribo-Zero Metabacteria

15.9 27.7 55.2

Ribo-Zero Metabacteria + Human/Mouse/Rat

4.9 26.7 56.3

- Cindi Hoover, JGI


What else about RiboZeroTM kit

• Outperformed other four tested kits/methods• Effective even on highly fragmented RNA sample • But needs sufficient input RNA (e.g. > 1 ug)• For environmental samples with very low RNA yield, no

rRNA depletion is the recommendation.

How about Archaea?

Giannoukos et al, Genome Biology 2012, 13:r23


Termite Hindgut Metatranscriptomics

- A case study

(Preliminary results)


Nasutitermes cornigerTermitidaeLaboratory colonyDry wood

Amitermes wheeleriTermitidaeSubtropical desertCow dung

Aim: Determine system-specific differences between termite species with different diets.

Termite samples in this study

Species:Family:Habitat:

Diet:


Overview of sequencing efforts

Nasutitermes Amitermes

(Lab colony)Dry wood

(Arizona desert)Cow dung

community analysis

16S pyrotag Metagenomics

Sanger at a QC level

454-titanium Metatranscriptomics

Illumina GAIIx – 1 x 34 bp

Illumina GAIIx – 2 x 76 bp

Illumina GAIIx – 2 x ll3 bp

1 lane

1 lane

1 lane

1 lane

1 lane

3 lanes


Bioinformatics workflow

- Edward Kirton, JGI


Summary

Metatranscriptomics is being advanced by next-generation sequencing technologies.

RiboZero kit is promising to knock down high rRNA content for more effective RNA-seq.

Bioinformatically removing rRNA reads should increase computational speed in de novo assembly, and improve the assembly of low-abundance mRNAs. Need to investigate algorithm that is more sensitive and computationally efficient to do this for large datasets.


• Phil Hugenholtz• Susannah Tringe• Edward Kirton• Kanwar Singh• Erika Lindquist• Feng Chen• Jeff Froula• Falk Warnecke• Natalia Ivanova• Martin Allgaier• Zhong Wang• Tao Zhang• Cindi Hoover• R&D group• Production group• Many others!

• Hans Peter Klenk

• Omri Wurtzel• Rotem Sorek

Acknowledgement

• Jose Escovar-Kousen

• Rudolph Scheffrahn

advancing science with dna sequence metatranscriptomics: challenges and progress shaomei he doe...

Documents

advancing science

dna sequence challenges

dna sequence validation

dna sequence objectives

raw rna

input rna

regulatory rna

length rrna